We have completed the description and sequencing of the exons/flanking regions of the UGT1 gene complex locus spanning some 350 kb on chromosome 2; its original version containing 6 exons 1 with each representing a gene was critical to our determining the first defects in patients with Crigler-Najjar(CN) diseases. The extension of the locus shows that in the 5' region each of thirteen different exons 1 with its own upstream promoter codes for the amino terminus of an isoform; the 13 exons 1, representing the UGT1A1-UGT1A13p genes, are arrayed in series with 4 common exons that code for identical carboxyl termini in the 3= region. This arrangement allows for the individually regulated synthesis of overlapping primary RNA transcripts such that the first exon in each is spliced to the 4 common exons. Because the last 7 exons 1 represented a highly homologous cluster, we employed 100-100 kb Bac/Pac clones to overlap gaps surrounding our previously selected cosmid clones. Although no gaps remain, we cannot be assured that all exons 1 have been detected. Exons 1A7-1A13P are 86-91% identical to each other and distantly related, 50-53 % identical, to the first six exons 1. Four genes are pseudo. The TATA box elements are either: TAATAA, AA(TA)7A, AAT9AAAT, or AT14AT. Promoter mutations for the UGT1A1 bilirubin gene have been uncovered in the genome of CN-I and CN-II individuals; sequence data showed a TA insertion at the AA(TA)7A box upstream of the bilirubin transferase gene. Transcriptional activity of a reporter gene fused to the 1A1 upstream region containing either a TA insertion or deletion was inhibited 80% and 35%, respectively, using HepG2 cells. Mobility shift assays with either radiolabeled AA(TA)6A, AA(TA)7A or AA(TA)8A showed that a hepatocyte-specific nuclear protein(s), at low (< 0.5 mg) concentrations, binds to the AA(TA)6A and AA(TA)8A mutant boxes but not to the wild type one. Additionally, the missense mutation in the CN-1 (R336W) or CN-II (I294T) patient was completely or partially inactivating, respectively. The viable mRNAs encoded at the UGT1 locus are differentially expressed. The expression of the UGT1A7-UGT1A10 cDNAs which we synthesized enabled the accumulation of a large body of substrate activity that suggests a distribution of proteins related to function in connection with chemical exposures. A conserved hydrophobic region was uncovered in the bilirubin UGT1A1 as a consequence of a deleterious mutation in a Crigler-Najjar (CN)Type I patient. The RAOARGOS computer program shows that this hydrophobic region and one in the phenol UGT1A6 are centered at position 170/171 over a di-phenylalanine and Tyr/Phe, respectively, with 1.22 and 1.14 positive factors compared to the threshold of 1.13 for such a structure; the steroid UGT2B7 isozyme lacks such a structure. Structure-function studies with UGT1A1 demonstrate that the Phe-170 is not replaceable whereas Leu can replace Phe-171. UGT1A6 metabolizes bilirubin at a low level; as bilirubin is highly hydrophobic, the 10-fold greater bilirubin glucuronidation with a much lower Km by UGT1A1 supports a critical role for the MRA in bilirubin glucuronidating activity.